This work reports on the direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase (DgAOR), a molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S] centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of the cases studied, electron transfer took place, although different redox reactions were responsible for the voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed.

Mossbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. Mossbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the Mossbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.

The dissimilatory nitrite reductase from Desulfovibrio desulfuricans ATCC 27774 catalyzes the reduction of nitrite to ammonia. Previous spectroscopic investigation revealed that it is a hexaheme cytochrome containing one high spin ferric heme and five low spin ferric hemes in the oxidized enzyme. The current study uses the high resolution of Mossbauer spectroscopy to obtain redox properties of the six heme groups. Correlating the Mossbauer findings with the EPR data reveals the pairwise spin-spin coupling among four of the heme groups. The other two hemes are found to be magnetically isolated. Reduction with dithionite and reaction with CO further indicate that only the high spin heme is capable of binding small exogenous ligands. These results confirm our previous finding that Desulfovibrio desulfuricans nitrite reductase contains six heme groups and that the high spin ferric heme is the substrate and inhibitor binding site.

In humans, the lymphomyeloid system has a fundamental role on iron metabolism promoting its recycling due to a continuous removal of effete red blood cells. Additionally, one of the most intriguing aspects of metalloporphyrins in biology is their effect on the immune system. However, the process of erythrocyte catabolism is still poorly understood and needs further research. In the present study, we attempt to investigate the nature and the possible physiologic role of Fe compounds released after erythrophagocytosis during the removal of red blood cells. Monocyte erythrophagocytosis in vitro experiments were done to characterize chemically the Fe compounds present inside the cells and in the culture supernatants. We tested the probable immunomodulatory functions of erythrophagocytosis products over lymphocyte cultures activated in vitro with T mitogens (alpha-CD3). Data obtained from atomic absorption spectroscopy confirmed the presence of Fe in the culture supernatants of monocyte cultures after erythrophagocytosis. Also, high-spin haem complexes derived from erythrocyte catabolism were detected by electron paramagnetic electronic resonance. Finally, in vitro activated lymphocyte proliferation experiments indicate the co-mitogenic properties of monocyte culture supernatants after red blood cells phagocytosis. Thus, the results of the present work provide evidence that culture monocyte supernatants after in vitro erythrophagocytosis contain Fe (III) high-spin haem complexes and show lymphocyte proliferation co-stimulatory properties.

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.

Metallothionein (MT) in the liver of gilthead seabreams (Sparus aurata L., 1758) exposed to Sado estuary (Portugal) sediments was quantified to assess the MT induction potential as a biomarker of sediment-based contamination by copper (Cu), cadmium (U), lead (Pb) and arsenic (As). Sediments were collected from two control sites and four sites with different levels of contamination. Sediment Cu, Cd, Pb, As, total organic matter (TOM) and fine fraction (FF) levels were determined. Generalized linear models (GLM) allowed integration of sediment parameters with liver Cu, Cd, Pb, As and MT concentrations. Although sediment metal levels were lower than expected, we relate NIT with liver Cd and also with interactions between liver and sediment Cu and between liver Cu and TOM. We suggest integrating biomarkers and environmental parameters using statistical models such as GLM as a more sensitive and reliable technique for sediment risk assessment than traditional isolated biomarker approaches. (C) 2007 Elsevier Inc. All rights reserved.

An air-stable formate dehydrogenase, an enzyme that catalyzes the oxidation of formate to CO2, was purified from a sulfate-reducing organism, Desulfovibrio desulfuricans ATCC 27774. The enzyme has a molecular mass of approximately 150 kDa (three different subunits: 88, 29 and 16 kDa) and contains three types of redox-active centers: four c-type hemes, nonheme iron arranged as two [4Fe-4S](2+/1+) centers and a molybdenum-pterin site. Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. Mo(V) EPR signals were observed in the native, reduced and formate-reacted states. EPR signals related to the presence of multiple low-spin hemes were also observed in the oxidized state. Upon reduction, an examination of the EPR data under appropriate conditions distinguishes two types of iron-sulfur centers, an [Fe-S] center I (g(max)=2.050, g(med)=1.947, g(min)=1.896) and an [Fe-S] center II (g(max)=2.071, g(med)=1.926, g(min)=1.865). Mossbauer spectroscopy confirmed the presence of four hemes in the low-spin state. The presence of two [4Fe-4S](2+/1+) centers was confirmed, one of these displaying very small hyperfine coupling constants in the +1 oxidation state. The midpoint redox potentials of the enzyme metal centers were also estimated.

The molybdenum EXAFS of the Mo(2Fe-2S) protein from Desulfovibrio gigas has been examined using fluorescence detection and synchrotron radiation. In the oxidized form the molybdenum environment is found to contain two terminal oxo groups and two long (2.47 A) Mo-S bonds. Evidence was also found for an oxygen or nitrogen donor ligand at 1.90 A. Addition of dithionite to the oxidized enzyme results in loss of a terminal oxo group, perhaps due to protonation. In addition, a 0.1 A contraction in the Mo-S bond lengths is observed. The behavior of both oxidized and dithionite-treated forms is similar to that observed previously with "desulfo" xanthine oxidase.

The gene encoding cytochrome c nitrite reductase (NrfA) from Desulfovibrio desulfuricans ATCC 27774 was sequenced and the crystal structure of the enzyme was determined to 2.3-A resolution. In comparison with homologous structures, it presents structural differences mainly located at the regions surrounding the putative substrate inlet and product outlet, and includes a well defined second calcium site with octahedral geometry, coordinated to propionates of hemes 3 and 4, and caged by a loop non-existent in the previous structures. The highly negative electrostatic potential in the environment around hemes 3 and 4 suggests that the main role of this calcium ion may not be electrostatic but structural, namely in the stabilization of the conformation of the additional loop that cages it and influences the solvent accessibility of heme 4. The NrfA active site is similar to that of peroxidases with a nearby calcium site at the heme distal side nearly in the same location as occurs in the class II and class III peroxidases. This fact suggests that the calcium ion at the distal side of the active site in the NrfA enzymes may have a similar physiological role to that reported for the peroxidases.

The dsr gene from Desulfovibrio gigas encoding the nonheme iron protein desulforedoxin was cloned using the polymerase chain reaction, expressed in Escherichia coli, and purified to homogeneity. The physical and spectroscopic properties of the recombinant protein resemble those observed for the native protein isolated from D. gigas. These include an alpha 2 tertiary structure, the presence of bound iron, and absorbance maxima at 370 and 506 nm in the UV/visible spectrum due to ligand-to-iron charge transfer bands. Low temperature electron paramagnetic resonance studies confirm the presence of a high-spin ferric ion with g values of 7.7, 5.7, 4.1, and 1.8. Interestingly, E. coli produced two forms of desulforedoxin containing iron. One form was identified as a dimer with the metal-binding sites of both subunits occupied by iron while the second form contained equivalent amounts of iron and zinc and represents a dimer with one subunit occupied by iron and the second with zinc.

A biosensor for nitrite analytical determination was developed using a cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desufuricans ATCC 27774 immobilized and electrically connected on a glassy carbon electrode by entrapment in an electrogencrated poly(pyrrole-viologen) matrix. The modified bioelectrode was studied by cyclic voltammetry and a catalytic current was observed in presence of nitrite. The linear range of the electrode response was 5.4-43.4 muM. The detection limit and the sensitivity were 5.4 muM and 1721 mA M-1 cm(-2), respectively. The K-M(app) value determined from the Lineweaver-Burk plot was 86 muM. The biosensor fully maintained its electroenzymatic activity towards nitrite after four days.. No catalytic response was observed in the presence of nitrate ions while interference from sulfites was considered negligible. Finally, the biosensor composition was optimized in term of monomer-enzyme ratio. (C) 2004 Elsevier B.V. All rights reserved.

The saturation magnetizations of the three iron cluster of ferredoxin II of Desulfovibrio gigas in both the oxidized and reduced states have been studied at fixed magnetic fields up to 4.5 tesla over the temperature range from 1.8 to 200 K. The low field (0.3 tesla) susceptibility of oxidized ferredoxin II obeys the Curie law over this entire temperature range. This establishes -2Jox greater than 200 cm-1 as the lower limit for the antiferromagnetic exchange coupling of oxidized ferredoxin II. The saturation magnetizations of reduced ferredoxin II at several fixed fields yield a nested family of curves which can be fit with spin S = 2 and D = -2.7(4) cm-1 (with E/D assigned the value 0.23 as determined by Mossbauer and EPR spectra). The low field susceptibility of reduced ferredoxin II also obeys the Curie law from approximately 4 up to 200 K. This establishes -2Jred greater than 40 cm-1 as the lower limit for the antiferromagnetic coupling of reduced ferredoxin II.

The kinetic properties of the electron-transfer process between reduced Desulfovibrio vulgaris cytochrome c3 and D. vulgaris flavodoxin have been studied by anaerobic stopped-flow techniques. Anaerobic titrations of reduced cytochrome c3 with oxidized flavodoxin show a stoichiometry of 4 mol of flavodoxin required to oxidize the tetraheme cytochrome. Flavodoxin neutral semiquinone and oxidized cytochrome c3 are the only observable products of the reaction. At pH 7.5, the four-electron-transfer reaction is biphasic. Both the rapid and the slow phases exhibit limiting rates as the flavodoxin concentration is increased with respective rates of 73.4 and 18.5 s-1 and respective Kd values of 65.9 +/- 9.4 microM and 54.5 +/- 13 microM. A biphasic electron-transfer rate is observed when the ionic strength is increased to 100 mM KCl; however, the observed rate is no longer saturable, and relative second-order rate constants of 5.3 x 10(5) and 8.5 x 10(4) M-1 s-1 are calculated. The magnitude of the rapid phase of electron transfer diminishes with the level of heme reduction when varying reduced levels of the cytochrome are mixed with oxidized flavodoxin. No rapid phase is observed when 0.66e(-)-reduced cytochrome c3 reacts with an approximately 25-fold molar excess of flavodoxin. At pH 6.0, the electron-transfer reaction is monophasic with a limiting rate of 42 +/- 1.4 s-1 and a Kd value of approximately 8 microM. Increasing the ionic strength of the pH 6.0 solution to 100 microM KCl results in a biphasic reaction with relative second-order rate constants of 5.3 x 10(5) and 1.1 x 10(4) M-1 s-1. Azotobacter vinelandii flavodoxin reacts with reduced D. vulgaris cytochrome c3 in a slow, monophasic manner with limiting rate of electron transfer of 1.2 +/- 0.06 s-1 and a Kd value of 80.9 +/- 10.7 microM. These results are discussed in terms of two equilibrium conformational states for the cytochrome which are dependent on the pH of the medium and the level of heme reduction [Catarino et al. (1991) Eur. J. Biochem. 207, 1107-1113].

The kinetic properties of the electron-transfer process between reduced Desulfovibrio vulgaris cytochrome c(3) and D. vulgaris flavodoxin have been studied by anaerobic stopped-flow techniques. Anaerobic titrations of reduced cytochrome c(3) with oxidized flavodoxin show a stoichiometry of 4 mol of flavodoxin required to oxidize the tetraheme cytochrome. Flavodoxin neutral semiquinone and oxidized cytochrome c(3) are the only observable products of the reaction. At pH 7.5, the four-electron-transfer reaction is biphasic. Both the rapid and the slow phases exhibit limiting rates as the flavodoxin concentration is increased with respective rates of 73.4 and 18.5 s(-1) and respective K-d values of 65.9 +/- 9.4 mu M and 54.5 +/- 13 CIM. A biphasic electron-transfer rate is observed when the ionic strength is increased to 100 mM KCl; however, the observed rate is no longer saturable, and relative second-order rate constants of 5.3 X 10(5) and 8.5 x 10(4) M(-1) s(-1) are calculated. The magnitude of the rapid phase of electron transfer diminishes with the level of heme reduction when varying reduced levels of the cytochrome are mixed with oxidized flavodoxin. No rapid phase is observed when 0.66e(-)-reduced cytochrome c(3) reacts with an similar to 25-fold molar excess of flavodoxin. At pH 6.0, the electron-transfer reaction is monophasic with a limiting rate of 42 +/- 1.4 s(-1) and a Kd value of similar to 8 mu M. Increasing the ionic strength of the pH 6.0 solution to 100 mu M KCl results in a biphasic reaction with relative second-order rate constants of 5.3 x 10(5) and 1.1 x 10(4) M(-1) s(-1) Azotobacter vinelandii flavodoxin reacts with reduced D. vulgaris cytochrome cs in a slow, monophasic manner with limiting rate of electron transfer of 1.2 +/- 0.06 s(-1) and a K-d value of 80.9 +/- 10.7 mu M. These results are discussed in terms of two equilibrium conformational states for the cytochrome which are dependent on the pH of the medium and the level of heme reduction [Catarino et al. (1991) Eur. J. Biochem. 207, 1107-1113].

Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (N(2)OR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of N(2)OR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica N(2)OR, or an electrostatic nature, in the case of P. denitrificans N(2)OR and A. cycloclastes N(2)OR. A set of well-conserved residues on the N(2)OR surface were identified as being part of the electron transfer pathway from the redox partner to N(2)OR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica N(2)OR sequence). Moreover, we built a model for Wolinella succinogenes N(2)OR, an enzyme that has an additional c-type-heme-containing domain. The structures of the N(2)OR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the N(2)OR domain is similar to that found in the other electron transfer complexes.

The multicopper enzyme nitrous oxide reductase (N 2OR) catalyzes the final step of denitrification, the two-electron reduction of N 2O to N 2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c 552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c 552, the reaction rate is dependent on the ET reaction and independent of the N 2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c 552 concentration dependence, we estimate the following kinetic parameters: K m c 552 = 50.2 +/- 9.0 muM and V max c 552 = 1.8 +/- 0.6 units/mg. The N 2O concentration dependence indicates a K mN 2 O of 14.0 +/- 2.9 muM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c 552 is used as the electron donor (p K a = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by (1)H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c 552 is placed near a hydrophobic patch located around the CuA center.

This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing to the environmental significance of the reaction it catalyzes, reduction of nitrous oxide, and the unusual nature of its catalytic center, named CuZ. The structure of the CuZ center, the unique tetranuclear copper center found in this enzyme, opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ center, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction. In addition, the determination of the structure of the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by theoretical calculations. The current knowledge of the structure, function, and spectroscopic characterization of the CuZ center is described here. We would like to stress that although many questions have been answered, the CuZ center remains a scientific challenge, with many hypotheses still being formed.

The final step of bacterial denitrification, the two-electron reduction of N(2)O to N(2), is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting non-active state (1Cu(II)/3Cu(I)) to the fully reduced state (4Cu(I)), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N(2)O allowed the identification of a new reactive intermediate of the catalytic centre, CuZ degrees , in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 +/- 0.9) x 10(5) M(-1 )s(-1) was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZ degrees species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/inactivation mechanism of the enzyme are discussed.